TY - JOUR
T1 - Mechanistic insights into human pre-mRNA splicing of human ultra-short introns
T2 - Potential unusual mechanism identifies G-rich introns
AU - Sasaki-Haraguchi, Noriko
AU - Shimada, Makoto K.
AU - Taniguchi, Ichiro
AU - Ohno, Mutsuhito
AU - Mayeda, Akila
N1 - Funding Information:
We are grateful to Drs. M. Yoshida and A. Malhotra for providing the spliceostatin A and the purified recombinant RNase R, respectively. We thank Drs. A.R. Krainer and R. Yoshimoto for their valuable suggestions, and members of our laboratory for their support and discussions. This work was funded by research grants from the Hori Information Science Promotion Foundation to N.S-H. and the Okawa Foundation for Information and Telecommunications to A.M.
PY - 2012/6/29
Y1 - 2012/6/29
N2 - It is unknown how very short introns (<65 nt; termed 'ultra-short' introns) could be spliced in a massive spliceosome (>2.7. MDa) without steric hindrance. By screening an annotated human transcriptome database (H-InvDB), we identified three model ultra-short introns: the 56-nt intron in the HNRNPH1 (hnRNP H1) gene, the 49-nt intron in the NDOR1 (NADPH dependent diflavin oxidoreductase 1) gene, and the 43-nt intron in the ESRP2 (epithelial splicing regulatory protein 2) gene. We verified that these endogenous ultra-short introns are spliced, and also recapitulated this in cultured cells transfected with the corresponding mini-genes. The splicing of these ultra-short introns was repressed by a splicing inhibitor, spliceostatin A, suggesting that SF3b (a U2 snRNP component) is involved in their splicing processes. The 56-nt intron containing a pyrimidine-rich tract was spliced out in a lariat form, and this splicing was inhibited by the disruption of U1, U2, or U4 snRNA. In contrast, the 49- and 43-nt introns were purine-rich overall without any pyrimidine-rich tract, and these lariat RNAs were not detectable. Remarkably, shared G-rich intronic sequences in the 49- and 43-nt introns were required for their splicing, suggesting that these ultra-short introns may recruit a novel auxiliary splicing mechanism linked to G-rich intronic splicing enhancers.
AB - It is unknown how very short introns (<65 nt; termed 'ultra-short' introns) could be spliced in a massive spliceosome (>2.7. MDa) without steric hindrance. By screening an annotated human transcriptome database (H-InvDB), we identified three model ultra-short introns: the 56-nt intron in the HNRNPH1 (hnRNP H1) gene, the 49-nt intron in the NDOR1 (NADPH dependent diflavin oxidoreductase 1) gene, and the 43-nt intron in the ESRP2 (epithelial splicing regulatory protein 2) gene. We verified that these endogenous ultra-short introns are spliced, and also recapitulated this in cultured cells transfected with the corresponding mini-genes. The splicing of these ultra-short introns was repressed by a splicing inhibitor, spliceostatin A, suggesting that SF3b (a U2 snRNP component) is involved in their splicing processes. The 56-nt intron containing a pyrimidine-rich tract was spliced out in a lariat form, and this splicing was inhibited by the disruption of U1, U2, or U4 snRNA. In contrast, the 49- and 43-nt introns were purine-rich overall without any pyrimidine-rich tract, and these lariat RNAs were not detectable. Remarkably, shared G-rich intronic sequences in the 49- and 43-nt introns were required for their splicing, suggesting that these ultra-short introns may recruit a novel auxiliary splicing mechanism linked to G-rich intronic splicing enhancers.
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U2 - 10.1016/j.bbrc.2012.05.112
DO - 10.1016/j.bbrc.2012.05.112
M3 - Article
C2 - 22640740
AN - SCOPUS:84862863639
SN - 0006-291X
VL - 423
SP - 289
EP - 294
JO - Biochemical and Biophysical Research Communications
JF - Biochemical and Biophysical Research Communications
IS - 2
ER -